Multiple and Essential Sp1 Binding Sites in the Promoter for Transforming Growth Factor-β Type I Receptorстатья из журнала
Аннотация: Maximal gene expression driven by the promoter for the transforming growth factor β type I receptor (TGF-βRI) occurs with a 1.0-kilobase pair fragment immediately upstream of exon 1. This region lacks a typical TATA box but contains CCAAT boxes, multiple Sp1, and PEBP2/CBFα binding sites among other possiblecis-acting elements. Alterations within two CCAAT box sequences do not mitigate reporter gene expression driven by the basal promoter, and no nuclear factor binds to oligonucleotides encompassing these sites. In contrast, other deletions or site-specific mutations reveal an essential Sp1 site in the basal promoter and several dispersed upstream Sp1 sites that contribute to maximal reporter gene expression. The proportions of transcription factors Sp1 and Sp3, and their ratios of binding to consensus elements, are maintained in bone cells at different stages of differentiation. Finally, nuclear factor that binds to PEBP2/CBFα-related cis-acting elements in the basal promoter sequence also occurs in osteoblasts. Our studies reveal that constitutive expression of TGF-βRI may be determined by constitutive nuclear factor binding to Sp1 sites, whereas other elements may account for the variations in TGF-βRI levels that parallel changes in bone cell differentiation or activity. Maximal gene expression driven by the promoter for the transforming growth factor β type I receptor (TGF-βRI) occurs with a 1.0-kilobase pair fragment immediately upstream of exon 1. This region lacks a typical TATA box but contains CCAAT boxes, multiple Sp1, and PEBP2/CBFα binding sites among other possiblecis-acting elements. Alterations within two CCAAT box sequences do not mitigate reporter gene expression driven by the basal promoter, and no nuclear factor binds to oligonucleotides encompassing these sites. In contrast, other deletions or site-specific mutations reveal an essential Sp1 site in the basal promoter and several dispersed upstream Sp1 sites that contribute to maximal reporter gene expression. The proportions of transcription factors Sp1 and Sp3, and their ratios of binding to consensus elements, are maintained in bone cells at different stages of differentiation. Finally, nuclear factor that binds to PEBP2/CBFα-related cis-acting elements in the basal promoter sequence also occurs in osteoblasts. Our studies reveal that constitutive expression of TGF-βRI may be determined by constitutive nuclear factor binding to Sp1 sites, whereas other elements may account for the variations in TGF-βRI levels that parallel changes in bone cell differentiation or activity. Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF, transforming growth factor; TGF-βR, transforming growth factor β receptor; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction.1The abbreviations used are: TGF, transforming growth factor; TGF-βR, transforming growth factor β receptor; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction. receptors occur on most cells, and a functional TGF-β type I receptor (TGF-βRI) is required for all known TGF-β-dependent effects. In some situations its activity is controlled by complex interactions with other cell surface components (1Massague J. Attisano L. Wrana J.L. Trends Cell Biol. 1994; 4: 172-178Abstract Full Text PDF PubMed Scopus (526) Google Scholar, 2Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1726) Google Scholar, 3Carcamo J. Zentella A. Massague J. Mol. Cell. Biol. 1995; 15: 1573-1581Crossref PubMed Google Scholar). However, in contrast to TGF-βRII and the cell surface proteoglycan also termed TGF-βRIII or betaglycan, expression of TGF-βRI is maintained on differentiated bone cells (4Centrella M. Casinghino S. Kim J. Pham T. Rosen V. Wozney J. McCarthy T.L. Mol. Cell. Biol. 1995; 15: 3273-3281Crossref PubMed Google Scholar). For these reasons, and because little is known about the molecular control of TGF-βRI expression, we cloned the rat TGF-βRI promoter and characterized several of its functional aspects in cultures of primary and continuous skeletal and nonskeletal cells derived from fetal rats. The rat TGF-βRI promoter lacks a typical TATA box, but initiates transcription at multiple sites within a 220-bp span upstream of the initial methionine codon in differentiated bone cells. The 3′-terminal 300-bp sequence encompassing this region contains a GC-rich CpG island, seven consensus Sp1 binding sites, and two CCAAT boxes. Transfection studies using different fragments of TGF-βRI promoter cloned upstream of the reporter gene luciferase demonstrated maximal activity by a 1.0-kb fragment that encompassed these and other possible cis-acting elements. Importantly, several dispersed elements appeared to cooperate for maximal reporter gene expression in osteoblast-enriched cultures (5Ji C. Casinghino S. McCarthy T.L. Centrella M. J. Cell. Biochem. 1996; 63: 478-490Crossref PubMed Scopus (29) Google Scholar). Coincident with this work, the human TGF-βRI promoter was cloned, and its sequence reveals a similar organization with identically spaced CCAAT box motifs (6Bloom B.B. Humphries D.E. Kuang P.-P. Fine A. Goldstein R.H. Biochim. Biophys. Acta. 1996; 1312: 243-248Crossref PubMed Scopus (57) Google Scholar).These features suggested that the TGF-βRI gene is driven by a constitutively active promoter that maintains expression of TGF-βRI in many cells. Nevertheless, this promoter is partly unusual to the extent that other promoters organized in a similar way tend to lack CCAAT box sequences. Imposed on this are our previous observations that the proportions of TGF-βRI mRNA and protein may vary with the osteoblast phenotype and that its levels are rapidly controlled by certain stimulatory and inhibitory bone growth regulators (4Centrella M. Casinghino S. Kim J. Pham T. Rosen V. Wozney J. McCarthy T.L. Mol. Cell. Biol. 1995; 15: 3273-3281Crossref PubMed Google Scholar,7Centrella M. McCarthy T.L. Canalis E. Mol. Cell. Biol. 1991; 11: 4490-4496Crossref PubMed Google Scholar). 2D. J. Chang, C. Ji, T. L. McCarthy, and M. Centrella, unpublished results.2D. J. Chang, C. Ji, T. L. McCarthy, and M. Centrella, unpublished results. Initial TGF-βRI promoter activity studies substantiate that osteoblast-related variations in steady state mRNA levels are controlled at least in part at the level of gene transcription (4Centrella M. Casinghino S. Kim J. Pham T. Rosen V. Wozney J. McCarthy T.L. Mol. Cell. Biol. 1995; 15: 3273-3281Crossref PubMed Google Scholar,5Ji C. Casinghino S. McCarthy T.L. Centrella M. J. Cell. Biochem. 1996; 63: 478-490Crossref PubMed Scopus (29) Google Scholar). 3C. Ji, D. J. Chang, T. L. McCarthy, and M. Centrella, unpublished results.3C. Ji, D. J. Chang, T. L. McCarthy, and M. Centrella, unpublished results. Therefore, the widespread expression of TGF-βRI, driven by a constitutively active promoter, may in some instances be regulated by othercis-acting regulatory elements.In the present study we investigated in more detail sequences within the TGF-βRI promoter that are required for maximal and basal activity. We examined the importance of two CCAAT boxes and various consensus and putative binding sites for Sp1 transcription factor family members that occur in this region and identified the presence of PEBP2/CBFα binding sites. Our results identify that some of elements are not used under basal conditions, some appear to be essential components of constitutive TGF-βRI gene expression, and yet others may help to determine phenotype-dependent TGF-βRI expression by differentiated bone cells.DISCUSSIONThe rat TGF-βRI promoter contains a variety ofcis-acting elements that could contribute to constitutive or conditional expression. By transfecting reporter gene constructs into osteoblast-enriched cultures, we previously defined regions within the TGF-βRI promoter that are associated with maximal and basal activity. To understand TGF-βRI gene expression in more detail, we have now defined certain important transcription factor binding sites that may control basal promoter activity in many cells.Similar to various growth factor receptor promoters, the TGF-βRI promoter lacks TATA box sequence but contains a GC-enriched so-called CpG island (22Gardiner-Garden M. Frommer M. J. Mol. Biol. 1987; 196: 261-282Crossref PubMed Scopus (2631) Google Scholar, 23Tazi J. Bird A. Cell. 1990; 60: 909-920Abstract Full Text PDF PubMed Scopus (366) Google Scholar) with many transcription factor Sp1 binding sites. Analogous to the human TGF-βRI promoter (6Bloom B.B. Humphries D.E. Kuang P.-P. Fine A. Goldstein R.H. Biochim. Biophys. Acta. 1996; 1312: 243-248Crossref PubMed Scopus (57) Google Scholar), CCAAT box-like sequences that occur in this region make them unlike promoters for many other growth factors or growth factor receptors (24Ishii S. Xu Y.-H. Stratton R.H. Roe B.A. Merlino G.T. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4920-4924Crossref PubMed Scopus (255) Google Scholar, 25Araki E. Shimada F. Uzawa H. Mori M. Ebina Y. J. Biol. Chem. 1987; 262: 16186-16191Abstract Full Text PDF PubMed Google Scholar, 26Sehgal A. Patil N. Chao M. Mol. Cell. Biol. 1988; 8: 3160-3167Crossref PubMed Scopus (222) Google Scholar, 27Cooke D.W. Bankert L.A. Roberts Jr., C.T. LeRoith B. Casella S.J. Biochem. Biophys. Res. Commun. 1991; 177: 1113-1120Crossref PubMed Scopus (74) Google Scholar, 28Saito H. Kouhara H. Kasayama S. Kishimoto T. Sato B. Biochem. Biophys. Res. Commun. 1992; 183: 688-693Crossref PubMed Scopus (32) Google Scholar, 29Pang J.-H.S. Hung R.-Y. Wu C.-J. Fang Y.-Y. Chau L.-Y. J. Biol. Chem. 1995; 270: 14123-14129Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 30Youussoufian H. Zon L.I. Orkin S.H. D'Andrea A.D. Lodish H.F. Mol. Cell. Biol. 1990; 10: 3675-3682Crossref PubMed Scopus (67) Google Scholar, 31Ye K. Dinarello C.A. Clark B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2295-2299Crossref PubMed Scopus (68) Google Scholar, 32Humphries D.E. Bloom B.B. Fine A. Goldstein R.H. Biochem. Biophys. Res. Commun. 1994; 203: 1020-1027Crossref PubMed Scopus (30) Google Scholar). Nonetheless, oligonucleotides spanning the CCAAT box sites do not bind detectable levels of nuclear protein, and reporter gene expression was not reduced when these sites were disrupted. Therefore, flanking sequences or the association of other transcription elements in nearby areas may limit the contribution of the CCAAT boxes to TGF-βRI expression under the conditions that we have examined so far.Unlike genes controlled by CCAAT box and TATA box elements, rat TGF-βRI mRNA transcription initiates from multiple locations, characteristic of a constitutively expressed gene controlled by an Sp1-dependent promoter (5Ji C. Casinghino S. McCarthy T.L. Centrella M. J. Cell. Biochem. 1996; 63: 478-490Crossref PubMed Scopus (29) Google Scholar, 33Briggs M.R. Kadonaga J.T. Bell S.P. Tjian R. Science. 1986; 234: 47-52Crossref PubMed Scopus (1055) Google Scholar). Although deletions that included various Sp1 binding sites invariably limited TGF-βRI promoter activity, some of these elements, clustered within a 0.3-kb sequence at the 3′ end of the promoter, appeared more essential than others. By gel shift and immunodetection assays, we determined that these regions associated to equivalent extents with either Sp1 or with the closely related transcription factor Sp3, also present in the rat cell nuclear extracts that we examined. This result is consistent with the similar structural features, conservation of DNA binding domains, and similar abilities of both Sp1 family members factors to recognize specific cis-acting elements with identical affinities (13Hagen G. Muller S. Beato M. Suske G. Nucleic Acids Res. 1992; 20: 5519-5525Crossref PubMed Scopus (522) Google Scholar, 14Dennig J. Hagen G. Beato M. Suske G. J. Biol. Chem. 1995; 270: 12737-12744Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 15Majello B. DeLuca P. Suske G. Lania L. Oncogene. 1995; 10: 1841-1848PubMed Google Scholar, 16Daniel S. Kim K.-H. J. Biol. Chem. 1996; 271: 1385-1392Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 17Sjottem E. Anderssen S. Johansen T. J. Virol. 1996; 70: 188-198Crossref PubMed Google Scholar, 34Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 35Hagen G. Dennig J. Preiss A. Beato M. Suske G. J. Biol. Chem. 1995; 270: 24984-24989Abstract Full Text Full Text PDF Scopus (186) Google Scholar). Unlike Sp1, Sp3 may reduce Sp1-dependent gene expression (34Hagen G. Muller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar). We detected two complexes reactive with antibody specific for Sp3 by gel shift analysis consistent with earlier reports (14Dennig J. Hagen G. Beato M. Suske G. J. Biol. Chem. 1995; 270: 12737-12744Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 15Majello B. DeLuca P. Suske G. Lania L. Oncogene. 1995; 10: 1841-1848PubMed Google Scholar, 16Daniel S. Kim K.-H. J. Biol. Chem. 1996; 271: 1385-1392Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Furthermore, whereas TGF-βRI and its mRNA levels vary by relation to other TGF-β receptors on various bone- and skin-derived cells, we did not find changes in the amounts of Sp1 or the ratio of Sp1 to Sp3 that could account for those differences in the various cell types, or in osteoblast-enriched cultures at various stages of differentiation. Consequently, in the basal state, constitutively low levels of TGF-βRI expression may be tempered by the presence of both Sp1 and Sp3.In other situations the proportions of Sp1 and Sp3 may change, or other cellular proteins may modify their function. For example, Sp1 forms heteromeric complexes with several cellular proteins. p107, a member of the retinoblastoma family of proteins, binds Sp1 and represses Sp1-dependent transcription, whereas retinoblastoma itself has been reported to interact with Sp1 and Sp3. Furthermore, p107 and retinoblastoma may complex with E2F, with cyclins, and cyclin-dependent kinases. Sp1 also interacts with the RelA subunit of transcription factor NF-κB, and the cellular protein YY1 (36Datta P.K. Raychaudhuri P. Bagchi S. Mol. Cell. Biol. 1995; 15: 5444-5452Crossref PubMed Scopus (91) Google Scholar, 37Udvadia A.J. Templeton D.J. Horowitz J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3953-3957Crossref PubMed Scopus (198) Google Scholar, 38Zerfass-Thome K. Schulze A. Zwerschke W. Vogt B. Mol. Cell. Biol. 1997; 17: 407-415Crossref PubMed Scopus (113) Google Scholar, 39Sif S. Capobianco A.J. Gilmore T.D. Oncogene. 1993; 7: 2501-2509Google Scholar, 40Seto E. Lewis B. Shenk T. Nature. 1993; 365: 462-464Crossref PubMed Scopus (247) Google Scholar). Therefore, several conditions may arise that could account for variations in Sp1 activity, and its ability to drive TGF-βRI gene expression during the cell cycle, in various cell lineages, or in cell phenotype development.Overall, our studies support a crucial role for several sequences throughout the TGF-βRI promoter that contain Sp1 binding sites. Deletion of 5′ upstream sequences, reducing the promoter region to 0.7 kb, decreases its activity by 40–60%. Elimination of either another 0.4 kb from the 5′ end or an internal downstream sequence further suppresses promoter function. However, elimination of 0.1 kb of sequence from the 3′ end, a region that itself directs only moderate reporter gene expression, potently suppresses the activity of longer promoter fragments that still retain multiple Sp1 binding sites. Therefore, several regions can contribute to optimal TGF-βRI gene expression, although sequence information within the 0.1-kb 3′ span is essential for basal promoter activity. Using several overlapping oligonucleotides spanning this region, we located a specific sequence where substitution by two nucleotides completely eliminated Sp1 binding and suppressed reporter gene expression from a minimal promoter fragment. These results confirm the importance of multiple Sp1 sites throughout the TGF-βRI promoter and establish that one downstream site at position −63 to −54, ∼90% homologous to consensus Sp1 binding sites, contributes heavily to basal promoter activity. This finding is analogous results with the TGF-α promoter, where several related but nonconsensus Sp1 binding sites are also required for optimal promoter activity (41Chen X. Wright K.L. Berkowitz E.A. Azizkhan J.C. Ting J.P. Lee D.C. Oncogene. 1994; 9: 3179-3187PubMed Google Scholar). It is difficult to compare our results directly with those for the human TGF-βRI promoter. Even the longest construct used to assess the human promoter region reached upstream only as far as 0.7 kb and, most importantly, did not contain the 3′ 109-bp sequence where we detect an essential Sp1 site (6Bloom B.B. Humphries D.E. Kuang P.-P. Fine A. Goldstein R.H. Biochim. Biophys. Acta. 1996; 1312: 243-248Crossref PubMed Scopus (57) Google Scholar). Thus, at least two regulatory sites, including the important downstream Sp1 binding site (numbered −80 to −71 in the human promoter), have not yet been assessed for their effect on gene expression driven by the human TGF-βRI promoter.Within the 3′-terminal 0.1-kb region of the TGF-βRI promoter, we also found a related binding site for members of the PEBP2/CBFα transcription factor family. PEBP2/CBFα family members (also termed polyoma virus enhancer binding protein 2, or PEBP2α; and acute myelogenous leukemia factors; Refs. 19Ito Y. J. Cancer Res. Clin. Oncol. 1996; 122: 266-274Crossref PubMed Scopus (30) Google Scholar, 20Hiebert S.W. Downing J.R. Lenny N. Meyers S. Curr. Top. Microbiol. Immunol. 1996; 221: 253-258Google Scholar, 21Wang S. Speck N. Mol. Cell. Biol. 1992; 12: 89-102Crossref PubMed Scopus (167) Google Scholar) were previously identified in nuclear extracts from differentiated osteoblasts and found to have a critical role in the expression of the osteoblast-related protein, osteocalcin (18Banerjee C. Hiebert S.W. Stein J.L. Lian J.B. Stein G.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4968-4973Crossref PubMed Scopus (146) Google Scholar, 42Merriman H.L. van Wijnene A.J. Hiebert S. Bidwell J.P. Fey E. Lian J. Stein J. Stein G.S. Biochemistry. 1995; 34: 13125-13132Crossref PubMed Scopus (218) Google Scholar, 43Geoffroy V. Ducy P. Karsenty G. J. Biol. Chem. 1995; 270: 30973-30979Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Our studies with the TGF-βRI have now identified a new target for PEBP2/CBFα activity beyond the virus-infected or immunological tissues where their identity was first established (19Ito Y. J. Cancer Res. Clin. Oncol. 1996; 122: 266-274Crossref PubMed Scopus (30) Google Scholar). The presence of a PEBP2/CBFα-related element in this downstream region of the TGF-βRI promoter may account in part for the high level of TGF-βRI mRNA and protein expression and promoter activity by differentiated osteoblasts (4Centrella M. Casinghino S. Kim J. Pham T. Rosen V. Wozney J. McCarthy T.L. Mol. Cell. Biol. 1995; 15: 3273-3281Crossref PubMed Google Scholar), imposed upon the constitutive levels regulated by Sp1 and other basal elements. We are continuing to characterize this and several other even more potent PEBP2/CBFα binding sites further upstream (see Fig. 1) to identify the osteoblast-enriched PEBP2/CBFα family members that bind to these sequences and to examine variations in PEBP2/CBFα expression during osteoblast differentiation.2,3In addition to the Sp1 and PEBP2/CBFα-related complexes, others designated as band U also form with an oligonucleotide from this important 3′-terminal control region. This sequence contains elements for two other transcriptional regulators, HNF-5 and heat shock protein 70 (Hsp70). By relative migration, the slower migrating band presently seems inconsistent with a complex containing theMr of transcription factor HNF-5. It also seems unlikely to be accounted for by Hsp70 because of the basal growth conditions of our studies and the presence of another possible Hsp70 site in oligonucleotide SX1 that does not exhibit the same complex. However, it may represent a complex containing a basal transcription factor of the TFII family. TFII-related proteins are commonly involved in the expression of many genes transcribed by polymerase II, although the TGF-βRI promoter lacks a TATA box where these agents customarily bind (5Ji C. Casinghino S. McCarthy T.L. Centrella M. J. Cell. Biochem. 1996; 63: 478-490Crossref PubMed Scopus (29) Google Scholar, 6Bloom B.B. Humphries D.E. Kuang P.-P. Fine A. Goldstein R.H. Biochim. Biophys. Acta. 1996; 1312: 243-248Crossref PubMed Scopus (57) Google Scholar). Nevertheless, our studies demonstrate that basal gene expression from the TGF-βRI promoter relies heavily on several Sp1 binding sites. One of these cis-acting elements, which occurs far downstream, appears essential for optimal TGF-βRI promoter activity. However, the effectiveness of these sites may be modified by other negative or positive transcription regulators whose expression may vary with cell phenotype or with other extracellular circumstances. These and other differences may account for changes in TGF-βRI levels and therefore sensitivity to this important growth regulator during development, differentiation, or hormonal control in skeletal tissue (4Centrella M. Casinghino S. Kim J. Pham T. Rosen V. Wozney J. McCarthy T.L. Mol. Cell. Biol. 1995; 15: 3273-3281Crossref PubMed Google Scholar, 44Centrella M. Rosen V. Horowitz M.C. Wozney J.M. McCarthy T.L. Bikle D.D. Negro-Vilar A. Hormonal Regulation of Bone Mineral Metabolism. Endocrine Society, Bethesda, MD1995: 211-226Google Scholar). Transforming growth factor-β (TGF-β) 1The abbreviations used are: TGF, transforming growth factor; TGF-βR, transforming growth factor β receptor; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction.1The abbreviations used are: TGF, transforming growth factor; TGF-βR, transforming growth factor β receptor; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction. receptors occur on most cells, and a functional TGF-β type I receptor (TGF-βRI) is required for all known TGF-β-dependent effects. In some situations its activity is controlled by complex interactions with other cell surface components (1Massague J. Attisano L. Wrana J.L. Trends Cell Biol. 1994; 4: 172-178Abstract Full Text PDF PubMed Scopus (526) Google Scholar, 2Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1726) Google Scholar, 3Carcamo J. Zentella A. Massague J. Mol. Cell. Biol. 1995; 15: 1573-1581Crossref PubMed Google Scholar). However, in contrast to TGF-βRII and the cell surface proteoglycan also termed TGF-βRIII or betaglycan, expression of TGF-βRI is maintained on differentiated bone cells (4Centrella M. Casinghino S. Kim J. Pham T. Rosen V. Wozney J. McCarthy T.L. Mol. Cell. Biol. 1995; 15: 3273-3281Crossref PubMed Google Scholar). For these reasons, and because little is known about the molecular control of TGF-βRI expression, we cloned the rat TGF-βRI promoter and characterized several of its functional aspects in cultures of primary and continuous skeletal and nonskeletal cells derived from fetal rats. The rat TGF-βRI promoter lacks a typical TATA box, but initiates transcription at multiple sites within a 220-bp span upstream of the initial methionine codon in differentiated bone cells. The 3′-terminal 300-bp sequence encompassing this region contains a GC-rich CpG island, seven consensus Sp1 binding sites, and two CCAAT boxes. Transfection studies using different fragments of TGF-βRI promoter cloned upstream of the reporter gene luciferase demonstrated maximal activity by a 1.0-kb fragment that encompassed these and other possible cis-acting elements. Importantly, several dispersed elements appeared to cooperate for maximal reporter gene expression in osteoblast-enriched cultures (5Ji C. Casinghino S. McCarthy T.L. Centrella M. J. Cell. Biochem. 1996; 63: 478-490Crossref PubMed Scopus (29) Google Scholar). Coincident with this work, the human TGF-βRI promoter was cloned, and its sequence reveals a similar organization with identically spaced CCAAT box motifs (6Bloom B.B. Humphries D.E. Kuang P.-P. Fine A. Goldstein R.H. Biochim. Biophys. Acta. 1996; 1312: 243-248Crossref PubMed Scopus (57) Google Scholar). These features suggested that the TGF-βRI gene is driven by a constitutively active promoter that maintains expression of TGF-βRI in many cells. Nevertheless, this promoter is partly unusual to the extent that other promoters organized in a similar way tend to lack CCAAT box sequences. Imposed on this are our previous observations that the proportions of TGF-βRI mRNA and protein may vary with the osteoblast phenotype and that its levels are rapidly controlled by certain stimulatory and inhibitory bone growth regulators (4Centrella M. Casinghino S. Kim J. Pham T. Rosen V. Wozney J. McCarthy T.L. Mol. Cell. Biol. 1995; 15: 3273-3281Crossref PubMed Google Scholar,7Centrella M. McCarthy T.L. Canalis E. Mol. Cell. Biol. 1991; 11: 4490-4496Crossref PubMed Google Scholar). 2D. J. Chang, C. Ji, T. L. McCarthy, and M. Centrella, unpublished results.2D. J. Chang, C. Ji, T. L. McCarthy, and M. Centrella, unpublished results. Initial TGF-βRI promoter activity studies substantiate that osteoblast-related variations in steady state mRNA levels are controlled at least in part at the level of gene transcription (4Centrella M. Casinghino S. Kim J. Pham T. Rosen V. Wozney J. McCarthy T.L. Mol. Cell. Biol. 1995; 15: 3273-3281Crossref PubMed Google Scholar,5Ji C. Casinghino S. McCarthy T.L. Centrella M. J. Cell. Biochem. 1996; 63: 478-490Crossref PubMed Scopus (29) Google Scholar). 3C. Ji, D. J. Chang, T. L. McCarthy, and M. Centrella, unpublished results.3C. Ji, D. J. Chang, T. L. McCarthy, and M. Centrella, unpublished results. Therefore, the widespread expression of TGF-βRI, driven by a constitutively active promoter, may in some instances be regulated by othercis-acting regulatory elements. In the present study we investigated in more detail sequences within the TGF-βRI promoter that are required for maximal and basal activity. We examined the importance of two CCAAT boxes and various consensus and putative binding sites for Sp1 transcription factor family members that occur in this region and identified the presence of PEBP2/CBFα binding sites. Our results identify that some of elements are not used under basal conditions, some appear to be essential components of constitutive TGF-βRI gene expression, and yet others may help to determine phenotype-dependent TGF-βRI expression by differentiated bone cells. DISCUSSIONThe rat TGF-βRI promoter contains a variety ofcis-acting elements that could contribute to constitutive or conditional expression. By transfecting reporter gene constructs into osteoblast-enriched cultures, we previously defined regions within the TGF-βRI promoter that are associated with maximal and basal activity. To understand TGF-βRI gene expression in more detail, we have now defined certain important transcription factor binding sites that may control basal promoter activity in many cells.Similar to various growth factor receptor promoters, the TGF-βRI promoter lacks TATA box sequence but contains a GC-enriched so-called CpG island (22Gardiner-Garden M. Frommer M. J. Mol. Biol. 1987; 196: 261-282Crossref PubMed Scopus (2631) Google Scholar, 23Tazi J. Bird A. Cell. 1990; 60: 909-920Abstract Full Text PDF PubMed Scopus (366) Google Scholar) with many transcription factor Sp1 binding sites. Analogous to the human TGF-βRI promoter (6Bloom B.B. Humphries D.E. Kuang P.-P. Fine A. Goldstein R.H. Biochim. Biophys. Acta. 1996; 1312: 243-248Crossref PubMed Scopus (57) Google Scholar), CCAAT box-like sequences that occur in this region make them unlike promoters for many other growth factors or growth factor receptors (24Ishii S. Xu Y.-H. Stratton R.H. Roe B.A. Merlino G.T. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4920-4924Crossref PubMed Scopus (255) Google Scholar, 25Araki E. Shimada F. Uzawa H. Mori M. Ebina Y. J. Biol. Chem. 1987; 262: 16186-16191Abstract Full Text PDF PubMed Google Scholar, 26Sehgal A. Patil N. Chao M. Mol. Cell. Biol. 1988; 8: 3160-3167Crossref PubMed Scopus (222) Google Scholar, 27Cooke D.W. Bankert L.A. Roberts Jr., C.T. LeRoith B. Casella S.J. Biochem. Biophys. Res. Commun. 1991; 177: 1113-1120Crossref PubMed Scopus (74) Google Scholar, 28Saito H. Kouhara H. Kasayama S. Kishimoto T. Sato B. Biochem. Biophys. Res. Commun. 1992; 183: 688-693Crossref PubMed Scopus (32) Google Scholar, 29Pang J.-H.S. Hung R.-Y. Wu C.-J. Fang Y.-Y. Chau L.-Y. J. Biol. Chem. 1995; 270: 14123-14129Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 30Youussoufian H. Zon L.I. Orkin S.H. D'Andrea A.D. Lodish H.F. Mol. Cell. Biol. 1990; 10: 3675-3682Crossref PubMed Scopus (67) Google Scholar, 31Ye K. Dinarello C.A. Clark B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2295-2299Crossref PubMed Scopus (68) Google Scholar, 32Humphries D.E. Bloom B.B. Fine A. Goldstein R.H. Biochem. Biophys. Res. Commun. 1994; 203: 1020-1027Crossref PubMed Scopus (30) Google Scholar). Nonetheless, oligonucleotides spanning the CCAAT box sites do not bind detectable levels of nuclear protein, and reporter gene expression was not reduced when these sites were disrupted. Therefore, flanking sequences or the association of other transcription elements in nearby areas may limit the contribution of the CCAAT boxes to TGF-βRI expression under the conditions that we have examined so far.Unlike genes controlled by CCAAT box and TATA box elements, rat TGF-βRI mRNA transcription initiates from multiple locations, characteristic of a constitutively expressed gene controlled by an Sp1-dependent promoter (5Ji C. Casinghino S. McC
Год издания: 1997
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: TGF-β signaling in diseases, Cell Adhesion Molecules Research, Bone Metabolism and Diseases
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